Maintenance of the stability of genetic information requires the accurate synthesis of DNA. In animal cells, DNA synthesis is performed by five distinct classes of DNA polymerase, alpha, beta, delta, epsilon and gamma. Our objective has been to characterize the accuracy of DNA synthesis by each of these enzymes and to analyze the errors committed by each in an attempt to understand how mutation rates are controlled. A major focus during the past year has been examination of the fidelity of the three putative replicative DNA polymerases, alpha, beta and epsilon. Accomplishments include the following. We have determined of the detailed error specificity of the four-subunit DNA polymerase alpha-DNA primase complex purified from yeast, designated Pol I. The analysis forms the basis for future work on this replicative polymerase, which is already in progress with less- (as well as more-) complicated forms of the enzyme. This study has also led to testable models for production of large and complex deletion errors and for frameshift errors at non-reiterated nucleotide positions. We have characterized a mismatch-specific exonuclease associated with a second polymerase from yeast, Pol II (the putative analogue to mammalian pol epsilon). We have examined the error specificity of DNA polymerase epsilon, which contains an associated proofreading exo- nuclease activity, to determine the contribution of base-selectivity and proofreading to fidelity. Parallel studies have begun with DNA polymerase delta, which also contains an associated exonuclease that may serve a proofreading function. Each of these enzyme characterizations is central to our attempts to describe the molecular details for the accurate replication of human genetic information. In order to better understand the effects of known mutagens and carcinogens on the fidelity of DNA synthesis, we intend to extend these types of analyses to DNA substrates that contain defined lesions.